Project summary/abstract This grant application responds to Program Announcement Number PA-18-671 (Ruth L. Kirschstein NRSA Individual Predoctoral Fellowship) by proposing protein engineering and computational protein design strategies to target vulnerable epitopes of Dengue virus, focusing on the E dimer epitope (EDE) and the DIII A/G strand epitope. Dengue virus, comprising 4 distinct serotypes, is a growing health threat with ~390 million new infections yearly. Infection with one Dengue serotype confers lifelong protection against the same serotype, yet heterotypic infections are associated with severe disease as cross-reactive, non-neutralizing humoral immunity may exacerbate infection via antibody-dependent enhancement (ADE). An effective vaccine must thus elicit robust and broadly-neutralizing antibody responses to provide protection and minimize risk of ADE. We will characterize and define effects, namely binding valency, relevant to potent neutralization by antibodies targeting the EDE. Preliminary observations suggest that some EDE antibodies may be unable to bind the virus bivalently, which is contrary to the otherwise frequent observation that IgGs? gain their potency by binding viral capsids bivalently. We propose engineering synthetic diFabs ? two Fabs connected by DNA linkers ? to augment the reach of the IgG?s two binding moieties. We will characterize binding and neutralization potencies of diFabs with linkers of varying lengths to define conditions that afford bivalent binding and perhaps greater neutralization potency. Exploring the ability for bivalent engagement of Dengue virions and engineering molecules capable of doing so has the potential to lend insight into effective neutralization mechanisms. We will also develop and characterize epitope-focusing immunogens based on the DIII A/G strand and EDE. In a mouse model, we will determine the in vivo immunogenicity profiles of resurfaced DIII immunogens, which we have engineered to maintain the A/G strand epitope and eliminate loops known to be associated with narrow or non-neutralizing responses. Neutralization activity, breadth, and relative contributions of cross-reactive or serotype-specific responses, will be determined. As soluble E does not readily dimerize, we will also engineer a single-chain E dimer immunogen via a computational rewiring method. Further resurfacing will be completed via a data-driven approach, using antibodies known to be associated with protective/enhancing responses to identify regions to be eliminated via mutagenesis while maintaining the EDE. In sum, the components of this proposal have the potential to contribute to our knowledge of neutralizing responses to Dengue thus advancing therapeutic and vaccine development. During this project, the PI Jennifer Lai will be trained in protein engineering and computational protein design methods. Notably, the PI will have the opportunity to carry out various stages ? in silico design, in vitro/in vivo evaluations of antigenicity/immunogenicity ? of vaccine development. Mentorship within a collegial environment will enable the PI?s professional development towards a career as an independent investigator and educator.